Allogeneic islets transplanted into the liver have shown promise clinically for treatment of Type 1 Diabetes (T1D), yet their supply is limited. These limitations have led to the investigation of human pluripotent stem cells (hPSC) as an unlimited source of functional ?-cells. Multiple investigators have demonstrated the feasibility of differentiating hPSC to immature ?-cells in vitro and successfully transplanted these cells into rodents which allowed further maturation into glucose-responsive insulin-producing ?-cells. The main challenges of deriving ?-cells in vitro from hPSCs for transplantation are i) the efficiency and consistency of the hPSC differentiation, and ii) previous transplants are typically performed at non-translatable sites, and the adaptation to clinically translatable sites adds additional inefficiencies. Herein, we propose an innovative strategy of culturing the hPSCs on microporous polymer scaffolds as a platform to obtain efficient differentiation of hPSCs to islet organoids in vitro, which contain multiple endocrine cell types that are found within an islet. Furthermore, the organoids can be directly transplanted on scaffolds at a clinically relevant site, namely the peritoneal fat, without disrupting the niche that develops within the pores. PI Dr. Shea has developed the scaffolds for the transplantation of primary islets into mice at a clinically translatable site that allows for efficient engraftment and function, and the reversal of hyperglycemia with a minimal islet mass. co-PI Dr. Spence is a developmental biologist with expertise in organoid culture that is collaborating on the scaffold design and analysis of in vivo maturation. Aim 1 will test the hypothesis that the differentiation of hPSC-derived pancreatic progenitors on 3D microporous scaffolds can increase the efficiency for forming islet organoids in vitro. Scaffolds will be created with controlled architecture and modified with extracellular matrix (ECM) proteins to facilitate organization and differentiation of cells into islet structures. The maturity of the organoids and cellular subpopulations will be monitored through flow cytometry, gene expression of pancreatic makers, the activity of key transcription factors, and insulin secretion. Established conditions for differentiating hPSC to ?-cells will be used as a control. Aim 2 will investigate the in vivo maturation and function following transplantation of scaffolds with islet organoids in the pores. We will investigate the survival and maturation of the organoids upon transplantation into the peritoneal fat, considered a translatable site, and observe the restoration of euglycemia in diabetic mouse models. Dr. Jan Stegemann will consult on vascularization of the graft. In collaboration with a leading islet biologist, Dr. Peter Arvan, we will assess the function of the transplanted islet organoids relative to that of native islets, and analyze the cells by flow cytometry and gene expression for relevant pancreatic markers and sub-populations that are observed. Collectively, these studies will develop scaffolds as a platform that can facilitate manufacturing of the organoids, which can be readily transplanted while maintaining the niche that maximally supports engraftment and function.